Oral administration of melanin for protection against radiation

ABSTRACT

Methods and compositions are provided for alleviating and/or preventing one or more side effects associated with exposure to radiation in a subject exposed to radiation or at risk for exposure to radiation comprising oral administration to the subject of an amount of an edible source of melanin effective to alleviate a side effect associated with radiation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/454,242 filed Mar. 18, 2011, the contents of which are herebyincorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAI51519, AI087625, AI52733-07 and S10RR027308 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to use of melanin-containing substances,such as black mushroom-based food supplements, for oral administrationfor alleviating side effects associated with exposure to radiation suchas ionizing radiation.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparenthesis. Full citations for these references may be found at the endof the specification immediately preceding the claims. The disclosuresof these publications are hereby incorporated by reference in theirentireties into the subject application to more fully describe the artto which the subject application pertains.

Melanin is a high molecular weight pigment that is ubiquitous in natureand has a variety of biological functions (5). Melanins are found in allbiological kingdoms. These pigments are among the most stable,insoluble, and resistant of biological materials (6). Melanins can havedifferent structures depending on the biosynthetic pathway and precursormolecules. Some definitions of melanin have focused on chemical andphysical properties of melanins instead of defined structures (7).Melanins can be synthesized in the laboratory by chemical means or bymany living organisms. Melanins formed by the oxidative polymerizationof phenolic compounds are usually dark brown or black (6). However,melanins may have other colors as illustrated by the finding thatdopamine-derived melanin is reddish-brown. Fungi can make melanins fromat least two major biosynthetic pathways, employing the precursor1,8-dihydroxynapthalene (DHN melanin) or the oxidation of suitabletyrosine derivatives like dihydroxyphenylalanine (DOPA-melanin) (6). Thefungus C. neoformans can make melanins from a wide variety of phenoliccompounds which are oxidized by a laccase enzyme (8-10). Many fungiconstitutively synthesize melanin (11).

Every year 1.4 million people are diagnosed with cancer in the U.S. andhalf of them will undergo some form of radiation therapy in the courseof their disease. The availability of radioprotective compounds wouldalleviate the morbidity associated with the radiation exposure. Thedoses received by millions of patients during diagnostic radiologicalprocedures are also very high (the dose of a multi-slice cardiac CT scanis equal to the dose from 300 chest X-rays) and are of great concern aswell; thus such patients would also benefit from the affordable andeffective radioprotectors. There is also importance for public safety tohave radioprotective agents readily available in the event of a nuclearaccident or terrorist attack.

Radioprotective agents that could be given prior to, or even during,radiation exposure would be of significant value in alleviating the sideeffects associated with exposure to ionizing radiation. Currently thereare no FDA-approved radioprotectors. It would be extremely beneficialfor hundreds of millions of people to have access to food supplementsthat could fill the niche in the absence of radioprotective drugs.

Fungal melanins can function as energy transducing molecules capable ofcapturing high energy electromagnetic radiation and converting it intoan energy form that is useful to fungal cells (1). Furthermore, fungalmelanins can be effective shields against radiation; the efficacy ofradioprotection by melanins is dependent on their chemical compositionand spatial arrangement (2). In addition to free reactive radicalscavenging, radioprotection by melanins involves prevention of freeradical generation by Compton recoil electrons through gradual recoilelectron energy dissipation by the π-electron-rich melanin until thekinetic energy of recoil electrons becomes low enough to be trapped bystable free radicals present in the pigment (3). It has also been shownthat melanin-based nanoparticles protect bone marrow in mice subjectedto external whole body radiation or radioimmunotherapy (4).

The present invention addresses the need for radioprotectants in humansat risk for radiation exposure using melanin-based products.

SUMMARY OF THE INVENTION

The invention provides methods for alleviating and/or preventing one ormore side effects associated with exposure to radiation in a subjectexposed to radiation or at risk for exposure to radiation comprisingoral administration to the subject of an amount of an edible source ofmelanin effective to alleviate a side effect associated with radiation.

The invention also provides a method for increasing the survival rate ofa plurality of subjects exposed to an amount of radiation likely to killthe plurality of subjects, comprising oral administration to each of theplurality of subjects of an amount of an edible source of melanineffective to increase the survival rate of the plurality of subjectsexposed to the amount of radiation likely to kill the plurality ofsubjects.

The invention also provides edible sources of melanin packaged for oraladministration to a subject for alleviating and/or preventing one ormore side effects associated with exposure of the subject to radiation,wherein the edible source provides melanin in an amount equivalent to atleast 8 mg of purified melanin per kg of body weight of the subject.

The invention also provides a drinkable suspension of melanin packagedfor oral administration to a subject for alleviating and/or preventingone or more side effects associated with exposure of the subject toradiation, wherein the drinkable suspension comprises at least 500 mgmelanin in a volume of 500 mL or less.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Survival of CD1 mice receiving synthetic pheomelanin before 9 Gywhole body radiation dose. Three hours before receiving the whole bodydose of 9 Gy, the mice were given by oral gavage either 100 mg/kg bodyweight synthetic pheomelanin followed by 5 days of antibiotic support,or PBS followed by 5 days of antibiotic support, or PBS alone.AB—antibiotic support. 10 mice per group were used. P values were 0.001and 0.002 when the survival in pheomelanin group was compared with PBSand PBS plus antibiotics groups, respectively.

FIG. 2. Survival of CD1 mice after 9 Gy whole body radiation dose. Threehours before receiving the whole body dose of 9 Gy the mice were givenby oral gavage either 1 g/kg body weight of Auricolaria judae mushroomsuspended in PBS, or 1 g/kg body weight of Auricolaria judae mushroomfollowed by antibiotics support for 5 days, or PBS followed byantibiotics support for 5 days, or PBS alone. There were 5 mice in PBSalone and in PBS plus antibiotic support groups, and 6 mice in mushroomsand mushrooms plus antibiotics groups. The P value was 0.015 for bothmushrooms and mushrooms plus antibiotics when compared to PBS alone andto PBS plus antibiotics controls. Ab—antibiotics support, Mum—mushrooms.

FIG. 3A-3E. Chemical composition of melanins and appearance of melaninsfrom various sources and mushrooms used in the study: a) structure ofeumelanin oligomer; b) structure of pheomelanin oligomer; c) electronmicrograph of purified microbial melanin (melanin “ghosts”); d)synthetic melanin—eumelanin (black) on the left and pheomelanin (brown)on the right; e) edible mushrooms used in the study—Boletus edulis(white mushrooms) on the left and Auricularia auricula-judae (blackmushrooms) on the right.

FIG. 4A-4G. Physico-chemical characterization of black and whitemushrooms: a, b) EPR of dried mushrooms: a) black mushrooms; b) whitemushrooms; c-e) oxidative HPLC of melanin purified from black mushrooms:c) background solution; d) PDCA standard eluting at 8 min.; e) melaninfrom black mushrooms showing PDCA peak; f, g) results of DPPH assay forantioxidant presence: f) butylated hydroxyanisole (BHA) positivecontrol; g) methanol extracts from black and white mushrooms.

FIG. 5A-5H. Survival of irradiated CD-1 mice fed with black ediblemushrooms, blood counts in the surviving mice and histology of the GItract and bone marrow. Mice were divided into groups of 5-6 and fed 1g/kg body weight black mushroom suspension in PBS, or PBS alone, or 1g/kg white mushroom suspension, or 1 g/kg white mushroom suspensionsupplemented with 100 mg/kg synthetic melanin via gavage needle. Onehour after mushroom administration mice were irradiated with 9 Gy doseof Cs-137 radiation at a dose rate of 2.5 Gy/min. a) Kaplan-Meyersurvival curves. The experiment was performed twice and was terminatedat day 45; b) white blood cells counts; c) platelet counts; d-h) H&Estained slides with tissues from control and irradiated mice. Left,non-irradiated controls; middle, black mushroom group; right, whitemushroom supplemented with melanin. d) stomach, magnification ×400; e)LI, magnification 400; f) SI, magnification ×200; g) bone marrow,magnification ×400; h) spleen, magnification ×100.

FIG. 6A-6D. Survival and weight change in CD-1 mice ted with differentdoses of synthetic pheomelanin and/or antibiotics and irradiated with 9Gy gamma radiation at 2.5 Gy/min: a) mice fed with 0-100 mg/kg bodyweight pheomelanin; b) mice fed with 100 mg/kg pheomelanin followed byantibiotics for 5 days, or given PBS only, or given PBS followed byantibiotics for 5 days; c) combined results from a) and b); d) weightchange in irradiated groups modeled using linear regression.AB—antibiotics.

FIG. 7A-7C. Histological evaluation of the tissue in surviving micepost-irradiation with 9 Gy gamma radiation at 2.5 Gy/min: a) stomach,small intestine, large intestine, liver and bone marrow. Mice received100 mg/kg pheomelanin plus antibiotics (upper row); 75 mg/kg pheomelanin(middle row); 0 mg/kg plus antibiotics (lower row); b) tissues from amouse receiving 100 mg/kg pheomelanin plus antibiotics—focalmicroadenoma of the small intestine (left panel) and bone marrow (rightpanel); c) cecum of a single survivor in 0 mg/kg plus antibiotics group.The same region of the cecum is shown with magnification ×250 in theleft panel, ×400 in the middle panel and ×1,000 in the right panel Eachslide is a higher magnification of the same region. Magnification ×400in a) and b).

FIG. 8A-8B. Toxicity evaluation of microbial and synthetic eumelanin innon-irradiated CD-1 mice: a) body weight of mice fed with 15 mg/kgmicrobial or synthetic eumelanin; b) histology of GI organs from CD-1mice fed with microbial eumelanin and sacrificed 24 hr later: left,stomach; middle, small intestine; right, colon. Original magnification×400.

FIG. 9A-9H. Radiation effects in CD-1 mice fed with 15 mg/kg body weightmicrobial or synthetic eumelanin and irradiated with 9 Gy gammaradiation at 2.5 Gy/min: (a-f) histology of GI tract tissues obtainedfrom irradiated CD-1 mice sacrificed at 4 hr (a-c) and at 24 hr (d-f)post-irradiation: a) stomach, synthetic eumelanin group; b) stomach,microbial eumelanin group; c) stomach, PBS. Fewer apoptotic cells areseen in stomach tissue of microbial melanin fed mice than in syntheticeumelanin or PBS groups; d) colon, synthetic eumelanin group; e) colon,microbial eumelanin group; f) colon, PBS control group; g) cumulativeweight loss in CD-1 mice; h) survival of the irradiated mice. Originalmagnification ×400.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for alleviating one or more side effectsassociated with exposure to radiation in a subject exposed to radiationor at risk for exposure to radiation comprising oral administration tothe subject of an amount of an edible source of melanin effective toalleviate a side effect associated with radiation.

The invention also provides a method for increasing the survival rate ofa plurality of subjects exposed to an amount of radiation likely to killthe plurality of subjects, comprising oral administration to each of theplurality of subjects of an amount of an edible source of melanineffective to increase the survival rate of the plurality of subjectsexposed to the amount of radiation likely to kill the plurality ofsubjects. One skilled in the art will know from then literature theamount of radiation likely to kill the plurality of subjects. Forexample, for human beings the LD_(50/60d) (i.e. the dose that causes 50%mortality with 60 days of exposure) in humans from acute, whole bodyradiation exposure is in excess of 250 rad (2.5 Gy) and usuallyapproximately 400 to 500 rads (4-5 Gy).

Preferably, melanin is administered to the subject in an amountequivalent to at least 8 mg of purified melanin per kg of body weight ofthe subject. For example, melanin can be administered in an ediblesubstance, containing at least 10% melanin by dry weight, of at least 80mg of edible substance per kg of body weight of the subject. Forexample, melanin can be administered as at least 80 mg of dry mushroomsper kg of body weight of the subject, where the mushrooms contain atleast 10% melanin by dry weight. In an embodiment, the melanin isadministered in the form of a drinkable suspension. In an embodiment,the edible source of melanin comprises a drinkable suspension of melaninpackaged for oral administration to a subject for alleviating and/orpreventing one or more side effects associated with exposure of thesubject to radiation, wherein the drinkable suspension comprises atleast 500 mg melanin in a volume of at least 10 mL.

The invention also provides an edible source of melanin packaged fororal administration to a subject for alleviating and/or preventing oneor more side effects associated with exposure of the subject toradiation, wherein the edible source provides melanin in an amountequivalent to at least 8 mg of purified melanin per kg of body weight ofthe subject. In an embodiment, the edible source provides melanin in anamount equivalent to at least 9, 10, 15 or 20 mg of purified melanin perkg of body weight of the subject.

The invention also provides a drinkable suspension of melanin packagedfor oral administration to a subject for alleviating and/or preventingone or more side effects associated with exposure of the subject toradiation, wherein the drinkable suspension comprises at least 250 mgmelanin in a volume of at least 10 mL. In an embodiment, the drinkablesuspension comprises at least 500 mg melanin in a volume of at least 10mL. In an embodiment, the drinkable suspension comprises at least 560 mgmelanin in a volume of at least 10 mL. In an embodiment, the drinkablesuspension comprises the melanin in at least 25 mL, 50 mL, 75 mL, 100mL, 125 mL, 150 mL, 175 mL, 200 mL, 250, mL, 500 mL or 750 mL. In anembodiment, substantially all the melanin is in particulate form orsmaller. The drinkable suspension can be galenical.

The invention also provides a powderized form of melanin packaged formaking a drinkable suspension by dilution with a drinkable liquid. Thepowderized form of melanin may be packaged, for example in a sachet. Inan embodiment, the powderized form of melanin is formulated so as topermit, upon reconstitution with at least 10 mL, 25 mL, 50 mL, 75 mL,100 mL, 125 mL, 150 mL, 175 mL, 200 mL, 250, mL, 500 mL or 750 mL adrinkable suspension providing at least 8 mg of purified melanin per kgof body weight of the subject who will drink the drinkable suspension.In an embodiment, the powderized form of melanin is formulated so as topermit, upon reconstitution with at least 10 mL, 25 mL, 50 mL, 75 mL,100 mL, 125 mL, 150 mL, 175 mL, 200 mL, 250, mL, 500 mL or 750 mL adrinkable suspension providing at least 250 mg, 500 mg or 560 mgmelanin.

The melanin can be isolated or derived from a melanin-containingbiological source where melanin constitutes at least 10% of the dryweight of the biological source. Melanin can also be synthesizedchemically. Melanin can also be provided by administering amelanin-containing biological source that comprises at least 10% melaninper dry weight of the biological source. In an embodiment, the melaninis in a composition substantially free of fungal material.

The biological source can be, for example, a melanin-containing plant,cell, fungus or microorganism such as a bacterium. Preferred fungiinclude melanin-containing edible mushrooms, such as Auricolariaauricular-judae or Pleurotus cystidiosus. A chemical source for melanincan be auto- or catalytic-polymerization of certain phenolic compoundslike L-dopa.

The biological source can be grown in the presence of a melaninprecursor, such as, for example, one or more of L-dopa(3,4-dihydroxyphenylalanin), D-dopa, catechol, 5-hydroxyindole,tyramine, dopamine, tyrosine, cysteine, m-aminophenol, o-aminophenol,p-aminophenol, 4-aminocatechol, 2-hydroxyl-1,4-naphthaquinone,4-metholcatechol, 3,4-dihydroxynaphthalene, gallic acid, resorcinol,2-chloroaniline, p-chloroanisole, 2-amino-p-cresol,4,5-dihydroxynaphthalene, 1,8-dihydroxynaphthalene, 2,7-disulfonic acid,o-cresol, m-cresol, and p-cresol.

The melanin can comprise allomelanin, pheomelanin and/or eumelanin.Eumelanins are derived from the precursor tyrosine. Pheomelanin isderived from the precursors tyrosine and cysteine. Allomelanins areformed from nitrogen-free precursors such as catechol andl,8-dihydroxynaphthalenes. In one embodiment, the ratio of pheomelaninto eumelanin is at least 1:1. Preferably, the melanin contains divalentsulfur.

Preferably, one or more internal organs of the subject are protectedfrom radiation. Preferably, the organ that is protected is one or moreorgan selected from the group consisting of bone marrow, liver, spleen,kidneys, lungs, and gastrointestinal tract.

The side effect associated with radiation can be one or more. of nausea,vomiting, abdominal pain, diarrhea, dizziness, headache, fever,cutaneous radiation syndrome, low blood cell count, infection due to lowwhite blood cells, bleeding due to low platelets, anemia due to low redblood cells, or death. Preferably, the subject's chance of survival isincreased following exposure to radiation. In one embodiment, the doseof radiation received by the subject would be lethal to the subject inthe absence of radioprotection.

The subject can be any animal. Preferably the subject is a mammal andmore preferably a human.

The radiation can comprise ionizing radiation. Ionizing radiation is ofsufficiently high energy that it ionizes atoms. The radiation can be,for example, one or more of gamma radiation, x-ray radiation,bremsstrahlung radiation, ultraviolet radiation, and particulateradiation (e.g., α-radiation and β-radiation). The source of theradiation can be a medical isotope. In a preferred embodiment theionizing radiation is gamma radiation, α-radiation or β-radiation. In anpreferred embodiment, the radiation is from a man-made source ofradiation. For example, the source of the radiation can be radiationtherapy used for treatment of disease (such as radiotherapy), radiationfrom a medical imaging device (such as a CT scanner), radiation used forradiation surgery (e.g. stereotactic radiation surgery), a nuclearweapon, or a nuclear reactor, such as a nuclear reactor in a power plantor submarine or high-altitude radiation, e.g. as experienced incommercial or military flights or space flight. In an embodiment, thehigh-altitude radiation is natural ionizing radiation experienced ataltitudes in excess of 20,000 ft. The source of radiation can resultfrom a terrorist attack. Thus, a man-made source of radiation caninclude that resulting from natural radioactive isotopes, but as appliedin a man-made therapy, power source or device.

Subjects expected to benefit from the present invention include, but arenot limited to, the following. Every second patient in the U.S. who isdiagnosed with cancer (1.4 million people per year are diagnosed in theU.S.) will undergo some form of radiation therapy during the course oftheir disease. Another group of patients who will benefit are those whoundergo CT (computer tomography) scans. 72 million CT scans areperformed in the U.S. every year. There is growing concern about highdoses of radiation that many patients receive during those scans, whichare often recommended for them several times per year. The dose from onehigh resolution cardiac multi-slice CT scan is equivalent toapproximately 100-600 chest X-rays or over 3-years' worth of naturalbackground radiation. Yet another group of patients who can benefit fromthe present invention are people undergoing so-called stereotacticradiosurgery (done with Particle beam (proton)), or Cobalt-60 based(photon), or linear accelerator-based for conditions such asarteriovenous malformations, benign brain tumors, and functionaldisorders including trigeminal neuralgia, essential tremor, andParkinson's tremor/rigidity. Additional subject who could benefit fromthe present invention are frequent fliers and airline personnel whosedoses are known to exceed the annual limit for radiation occupationalworkers, nuclear medicine and radiology professionals, personnel at thenuclear power plants and nuclear reactors, and military personnel innuclear submarines, as well as victims of radiation accidents andterrorist attacks.

In an embodiment of the methods, the treatment results in reducing thelikelihood that the exposed subject will develop a cancer as a result ofchronic radiation exposure over an extended time period.

Melanin could be provided in the form of dry black mushrooms suspendedin palatable liquid (“melanin shakes”). The mushroom that could be usedinclude black edible mushrooms such as Auricularia auricular-judae.Mushrooms such as Auricularia auricular-judae can be grown as otheredible mushrooms in a basement of a building when provided with humidityand nutrients, dried, powderized and formulated into “melanin shakes” bymixing it with flavored water or fruit juice. Shakes with differentflavors can be made. The packaging can be standard individual juicecartons, e.g. 100 mL volume. Melanin could also be provided in otheredible forms, e.g., melanin brownies. Alternatively, naturally occurringor synthetic melanins can be isolated or synthesized, respectively, andadded to foodstuffs to create products suitable for oral ingestion. Innon-limiting embodiments, the melanin from black mushrooms can beprocessed so as to be particulate or powderized. In an embodiment, themelanin is from an organism, such as a fungi, which has been exposeditself to radiation in an amount effective to increase the melaninproduction in the organism (radiosynthesis). In an embodiment, theorganism has been grown under conditions comprising the presence of amelanin precursor. Methods for both radiosynthesis and growing in thepresence of a melanin precursor are described in U.S. Patent ApplicationPublication No. US 2009-0328258 A1, published Dec. 31, 2009, which ishereby incorporated by reference.

Also provided is a drinkable suspension of melanin packaged for oraladministration to a subject for alleviating and/or preventing one ormore side effects associated with exposure of the subject to radiation,wherein the drinkable suspension comprises at least 500 mg melanin in avolume of at least 10 mL. In an embodiment, the drinkable suspensioncomprises at least 500 mg melanin in a volume of at least 100 mL.

In an embodiment, the subject has been, is being, or will be exposed toa single radiation exposure of 10 mGy, 20 mGy, 50 mGy, 100 mGy, 500 mGy,1Gy, 1.5 Gy, 2Gy or greater, 5 Gy or greater, 7.5 Gy or greater, 10 Gyor greater or greater than 10 Gy. In humans, a whole-body exposure to 5or more Gy of high-energy radiation at one time usually leads to deathwithin 14 days.

In embodiments of the methods and compositions, including suspensions,the melanin is not in the form of melanized nanoparticles.

In an embodiment, the methods further comprise administering one or moreantibiotics to the subject.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

Experimental Details EXAMPLE 1

Radiation protection with synthetic melanin: The radioprotectiveproperties of fungal and synthetic melanins were tested by oraladministration of synthetic pheomelanin to mice before whole bodyexposure to. 1.5 lethal dose (9 Gy) of gamma radiation. The whole bodydose of 9 Gy is also 2.5 times the lethal dose for a human. Theresulting survival of mice protected with melanins (FIG. 1) providedencouragement for the use and development of melanin-based products asradioprotectants in humans at risk for radiation exposure.

Radiation protection with black edible mushrooms: In a follow-upexperiment, it was investigated whether the result with syntheticmelanin would apply to natural melanins. The edible fungus Auriculariaauricular-judae was selected since it is heavily melanized. Three hoursbefore receiving the whole body dose of 9 Gy, groups of 5-6 CD1 micewere given by oral gavage 1 g/kg body weight of Auriculariaauricular-judae mushroom suspended in PBS, or 1 g/kg body weight ofAuricularia auricular-judae mushroom followed by antibiotics support for5 days after irradiation, or PBS followed by antibiotics support for 5days after irradiation, or PBS alone. Mice were monitored for theirsurvival for 30 days since in radioprotection experiments mice areconsidered to be surviving indefinitely beyond that point. The resultsof the experiment are shown in FIG. 2. Black mushroom Auriculariaauricular-judae significantly prolonged the survival of lethallyirradiated mice, with 30% of mice given mushrooms alone or mushroomswith antibiotic support surviving for 30 days. There were 5 mice in PBSalone and in PBS plus antibiotic support groups, and 6 mice in mushroomsand mushrooms plus antibiotics groups. The P value was 0.015 for bothmushrooms and mushrooms plus antibiotics when compared to PBS alone andto PBS plus antibiotics controls.

Given that synthetic melanins and melanin in microscopic fungi have asimilar structure as the melanin found in edible mushrooms, a foodsupplement could be used to supply melanin in the form, for example, ofdry black mushrooms suspended in palatable liquid (“melanin shakes”) toindividuals to be subjected to radiation exposure. In studies with oraladministration of melanin the protective dose of purified melanin was100 mg/kg in a mouse, which will be 8 mg/kg purified melanin in a humantaking into consideration the different weight to body surface arearatios in mice and humans. Provided that melanin constitutes at least10% of a dry mushroom weight—in a mouse experiment described above, micereceived 1 g/kg of Auricularia auricular-judae which in a human will beequal to 80 mg/kg of dry mushrooms, or 5.6 g per 70 kg person.Auricularia auricular-judae can be grown as other edible mushrooms in abasement of a building when provided with humidity and nutrients, dried,powderized and formulated into “melanin shakes” by mixing it withflavored water or fruit juice. Shakes with different flavors can bemade. The packaging can be standard individual juice cartons, e.g. 100mL volume.

EXAMPLE 2

Melanin-containing edible mushrooms offered the highest degree ofradioprotection without antibiotic support. The radioprotective efficacyof melanin delivered as a natural food source was evaluated. The blackedible mushroom Auricularia auricula-judae (common names Jelly Ear orJudas Ear) was selected as a source of edible melanin and the whitemushroom Boletus edulis (common names porcino or bun bun) as amelanin-devoid control (FIG. 3E). Both types of mushroom arebasidiomycetes that are used in Western and Asian cuisines and areavailable commercially in dried form. The presence of melanin inAuricularia auricula-judae (black mushrooms) and its absence in Boletusedulis (white mushrooms) was demonstrated by electron paramagneticresonance (EPR) with characteristic melanin “signature” signal in blackmushrooms (FIG. 4A) and background only—in white mushrooms (FIG. 4B).Melanin purified from black mushrooms using the protocol developed inour laboratories (19) constituted approximately 10% of black mushroomsdry weight and was further characterized by elemental analysis andoxidative high performance liquid chromatography (HPLC). Eumelanins arecomposed of 5,6-dihydroxyindole (DHI) and5,6-dihydroxyindole-2-carboxylic acid (DHICA) monomer units with 6-9%nitrogen (20, 21). In parallel, fungi also synthesize eumelanin from1,8-dihydroxynaphthalene (DHN) via pentaketide synthetic pathway andsuch melanin does not contain nitrogen in its structure (22). Theelemental analysis determined that there was 44% carbon, 5% hydrogen and2% nitrogen in black mushroom melanin. The low percentage of nitrogensuggested that the pigment was primarily DHN-melanin, while the HPLC ofoxidized melanin gave additional information about its structure (FIG.4C-E). The presence of pyrrole-2,3-dicarboxylic acid (PDCA), which is anoxidation product of DHI-derived units in oxidized melanin allowed aconclusion that melanin in black mushrooms was a mixture of DHN and DHImelanins. In addition, it was considered whether mushroom-associatedantioxidants could contribute to the radioprotective effect and comparedthe antioxidant contents of black and white mushrooms using2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The DPPH is a stable freeradical having a deep violet color in solution. The radical scavengingactivity of a sample can be measured as a decolorizing effect followingthe trapping of the unpaired electron of DPPH12. There was no differencein soluble antioxidant content between black and white mushrooms (FIG.4F, 4G), thus excluding differences in antioxidants as the basis ofblack mushroom-mediated radiation protection in vivo. It is important tonote, that only soluble antioxidants are measured in this assay as they.are extracted into methanol. Melanin which also possesses powerful freeradical scavenging properties by virtue of being a stable free radical(24) cannot contribute to the results of this assay as it is not solublein methanol or any other common solvents.

To test the radioprotective properties of edible mushrooms in mice wefirst need to determine the time between feeding mice with mushrooms andirradiation to ascertain the presence of mushrooms in GI tract duringirradiation. The fluorescent imaging was performed by utilizing thenatural autofluorescence of white mushrooms. Mice were given mushroomsuspension with a gavage needle and imaged on IVIS Spectrum ImagingSystem at 15, 30 and 60 min post-feeding. 675/30 nm and 840/20 nmfilters were used for excitation and emission, respectively. Mice weregiven 1 g/kg body weight white mushrooms suspension in water via gavageneedle and imaged in supine position under Isoflurane anesthesia. Themushrooms were in the stomach at 15 and 30 min post-feeding and movedinto the intestines to the large extent at 60 min (data not shown). Asintestines are more sensitive to ionizing radiation than stomach, weselected 60 min as time to administer radiation in order to ensuremaximum protection for the most sensitive part of GI tract. Armed withthese results, we conducted a radioprotection study in mice given lethalwhole body dose of 9 Gy. Groups of 5-6 CD-1 mice were used in theexperiment which was then repeated with the similar results. Blackmushrooms were administered as suspension in sterile PBS via gavage as 1g/kg body weight. The control groups included mice given only sterilePBS, or white mushrooms as 1 g/kg body weight dose. To establish thatthe melanin pigment was indeed the radioprotective substance in blackmushrooms an additional group of mice was given white mushrooms as 1g/kg body weight dose supplemented with 100 mg/kg of synthetic melanindosed to match its contents in black mushrooms. All mice in the PBSgroup and 80% in the white mushrooms—fed group died by day 14 postirradiation (FIG. 5A). The remaining 20% of mice in the whitemushroom-treated group died by day 25. This trend toward prolongation insurvival in comparison with PBS alone group which was not statisticallysignificant (P=0.07) and might be explained by the presence ofantioxidants in white mushrooms which could have produced localprotective effects in the gut. Strikingly, in black mushrooms and inwhite mushrooms supplemented with synthetic melanin groups, 60 and 75%of mice survived (P=0.002 and 0.001), respectively, up to day 45 whenthe experiment was terminated to perform the histological examination oftheir tissues (FIG. 5A). At the same time the white blood cell counts inblack mushroom and in melanin-supplemented groups were not differentfrom the non-irradiated controls (P=0.06) (FIG. 5B), while plateletcounts were lower in both irradiated groups (P=0.03) (FIG. 5C), however,at the levels which ensure recovery in mice receiving radiationtreatment (25). In lethally irradiated mice mortality results fromdamage to rapidly dividing tissues such as GI mucosa (26) and bonemarrow suppression (27, 28). There were no signs of radiation damage inthe stomachs, large and small intestines (LI and SI, respectively) inthe surviving mice in black mushroom and melanin-supplemented groups(FIG. 5D-F). Bone marrow of irradiated mice had slight myeloidhyperplasia (FIG. 5G) and the spleens architecture was normal with someextramedullary hematopoesis (FIG. 5).

Synthetic pheomelanin in combination with antibiotics protected themajority of mice against lethal dose of radiation. It was hypothesizedthat high linear attenuation coefficient and stable radical contents(18) of the synthetic pheomelanins would translate into radioprotectionin mice. The dose response experiments demonstrated that orallyadministrated synthetic pheomelanin protected mice irradiated with 9 Gyat 2.5 Gy/min in a dose-dependent manner. Over a 40 day survival study,no protection was identified in mice receiving 25 mg/kg pheomelanin.Mice fed with 50 mg/kg had a 6.6% survival rate (P=0.02), while thosereceiving 75 mg/kg had a 20% survival rate (P=0.01) (FIG. 6A). However,no protection was observed when the pheomelanin dose was increased to100 mg/kg (P=0.06). The possibility that the lack of protection at thehigher melanin dose was a consequence of bowel-related effects thatpredisposed animals to bacterial sepsis was considered. Hence, in afollow-up study, the effect of antimicrobial therapy on survival afterpheomelanin administration and lethal irradiation was evaluated. Micewere given either PBS alone, or PBS in combination with antibiotics or100 mg/kg pheomelanin in combination with antibiotics which resulted in40 day survival of 0%, 16.7%, and 80% (P=0.005), respectively (FIG. 6B,6C).

The rate of weight loss in the different treatments groups was analyzedby linear regression (FIG. 6D). The highest weight loss was observed inthe groups given PBS alone or PBS in combination with antibiotics: a 1.6and 1.1% decrease in total body weight per day, respectively. Mice givenpheomelanin displayed significantly less weight loss when compared toPBS alone group: the group treated with 50 mg/kg of pheomelanin lost1.0% of their body weight per day (P=0.02), while the group treated with75 mg/kg had a 0.7% loss in body weight per day (P=0.015). Mostimportantly, mice receiving 100 mg/kg pheomelanin plus antibioticsactually gained weight at a rate of 1.0% per day after irradiation, adifference that was significant in comparison with all other groups(P<0.05).

Histological evaluation of the tissue in surviving mice confirmed thebody weight data by revealing no damage to the stomach, small intestine,large intestine, liver and bone marrow in surviving mice treated with100 mg/kg pheomelanin plus antibiotics (FIG. 7A, upper row) or 75 mg/kgpheomelanin (FIG. 7A, middle row). Among the surviving mice in the 100mg/kg pheomelanin plus antibiotics group (80% survival) only one mousehad a focal microadenoma in the small intestine (FIG. 7B, left panel)and moderately depleted bone marrow cellularity (FIG. 7B, right panel).The single survivor in PBS plus antibiotics group exhibited multifocallymphohistiocytic and plasmacytic periportal infiltrates in the liver(FIG. 7A, tower row) and a focal perforation in the cecum with chronicactive peritonitis (FIG. 7C).

Microbial and synthetic eumelanins prolonged survival of lethallyirradiated mice. Before carrying out irradiation studies we evaluatedwhether there was any toxicity associated with oral administration ofmicrobial and synthetic melanins to CD-1 mice. Measures of toxicity werethe body weight over 10 days and histological evaluation of gut tissue.Microbial and synthetic eu- and pheomelanins proved to be non-toxic withmice steadily gaining weight during the observation period which wasconfirmed by the normal histology of the gut (FIG. 8).

Encouraged by the lack of toxicity of microbial and synthetic melanins,the efficacy of orally administered synthetic and microbial eumelaninsin protecting CD-1 mice against lethal irradiation was evaluated.Histological evaluation of GI tissues obtained from mice 4 hrpost-irradiation with 9 Gy at 2.5 Gy/min from 137Cs source revealed thatmice fed with microbial eumelanin had approximately 40% fewer apoptoticcells in stomach tissue than mice fed synthetic eumelanin or PBS (FIG.10A-C). At 24 hr this trend continued with glandular cells being lessattenuated in stomachs of mice fed with microbial eumelanin than in micefed with synthetic eumelanin. Simultaneously, there were approximately25% more mitotic figures and less apoptotic cells in both eumelaninsgroups in comparison with control PBS fed mice. In the small intestinethere was no apparent difference between treatment groups. At 24 hr inthe colonic glands of mice fed microbial eumelanin there was 30% lesscellular reaction and apoptosis compared to the other colon samples(FIG. 9D-F).

For the first four days post-irradiation, mice fed with microbialeumelanin lost slightly less weight than mice fed either PBS orsynthetic eumelanin (FIG. 9G). By day 5, the cumulative weight loss inall groups had equalized and for the rest of the observation period theweight loss was the least pronounced in mice fed with syntheticeumelanin. The overall survival on day II post-irradiation was 100% insynthetic eumelanin group, 66%—in the microbial eumelanin group and33%—in control mice fed with PBS, with the last mouse in this groupdying on day 16 (FIG. 9J). For the duration of study, the mean survivalfor mice fed with microbial eumelanin was 13 d, for control PBS fedmice—12.7 days and for synthetic eumelanin group—19 days (P=0.01,Mandel-Cox test).

Discussion

There is an ongoing and urgent need for oral radioprotectors that areinexpensive, do not require refrigeration for storage and transportation(“cold chain”), and are suitable for distribution to large numbers ofpeople in the event of radiation emergencies such as the recent nuclearaccident at Fukushima-Daiichi nuclear plants. This need is enhanced bythe fact that many developing nations are considering increased relianceon nuclear power as an alternative to fossil fuels and that a majorexpansion in nuclear programs carries significant risks as evidenced bytwo major accidents at Chernobyl and Fukushima-Daiichi in the space ofone generation. One potential radioprotector that has been studiedextensively is amifostine (28-30). It belongs to the class of freeradical scavengers that includes aminothiols and phosphorothioates, andis administered as a prodrug that must be metabolized to an active formto be effective. While this drug has some radioprotective efficacy, italso has several undesirable properties, including a relatively lowradioprotective capacity, potentially serious side effects such asanaphylaxis and the need for intravenous administration. In a study byBurdelya et al. (31), a different approach to radioprotection was takenby pharmacologically suppressing apoptosis in the irradiated cells. Thiswas done by pre-treating experimental animals with flagellin-derivedpolypeptide which binds to Toll-like receptor 5 and activates nuclearfactor-κB signaling. While this method showed some promise, the drugalso has to be given parenterally and might have carcinogenic sideeffects by virtue of interfering with the process of apoptosis.

Herein, in vivo studies were conducted to evaluate protective effect ofdifferent types of orally administered melanins on the GI tract in micereceiving a lethal dose of 9 Gy at a high dose rate. The radioprotectiveeffects of melanin are proposed to be based on controlled dissipation ofCompton electron energy by melanin which results in a decreased numberof interactions between Compton electrons and cellular milieu and thescavenging of free reactive radicals by melanin (18). The protectiveeffects of two eumelanins—microbial eumelanin purified from C.neoformans and commercially available synthetic eumelanin were compared.Histological examination of the stomachs and colons of the irradiatedmice revealed that the mice given microbial eumelanin were betterprotected than those given synthetic eumelanin or controls. However,this early protective effect of microbial eumelanin did not extend intothe long-term protection while synthetic eumelanin administrationresulted in statistically significant prolongation in survival. Thissurprising observation may be explained by the inflammation whichmicrobial eumelanin can cause in the mucosa due to the persistentpresence of immunogenic proteins and polysaccharides intertwined withits structure even after rigorous multi-step purification (32). Melanin‘ghosts’ derived from melanized fungal cells contain cell wallcomponents which are known to be highly immunogenic. For example,zymosan particles prepared from yeast cell wall are notoriouslypro-inflammatory (33) and C. neoformans-derived melanin have been shownto trigger direct inflammation (34). Such inflammation might increasethe damage sustained from radiation and be accompanied by edema whichcould explain the less significant weight loss in the microbialeumelanin group in comparison with the synthetic eumelanin and controlgroups during the first four days after irradiation. The syntheticeumelanin afforded statistically significant prolongation in survivalfor the overall duration of experiment, which provided impetus forfurther investigation of its role in radioprotection by orallyadministering to mice synthetic pheomelanin which has higher number ofstable free radicals than eumelanin and was more radioprotective invitro (18).

Pheomelanin protected mice in a dose-dependent manner in the dose rangeof 25-75 mg/kg body weight. Since the 100 mg/kg dose did not protectmice, it was hypothesized that the higher dose of melanin may have hadunanticipated adverse effects in damaged tissue. To explore thecontribution of associated bacteremia to the mortality antibiotics wereadministered to mice post-irradiation. Antibiotic administrationresulted in 80% survival of irradiated mice treated with 100 mg/kgpheomelanin. When compared to published data—pheomelanin plusantibiotics was more protective then amifostine (60% survival after 9 Gydelivered at 1 Gy/min (17)), and equal to flagellin-derived polypeptide(80% survival after 9 Gy delivered at 2.3 Gy/min (20)). The increase inradiation dose rate is known to make the cellular repair mechanisms lessefficient (35). The histological evaluation of the surviving mice ingroups protected with pheomelanin alone or with pheomelanin andantibiotics revealed no obvious radiation damage to the major organs.Among the survivors in the group receiving 100 mg/kg pheomelanin plusantibiotics only one mouse had any abnormality in its major organs,which consisted of a moderate depletion of the bone marrow and anisolated microadenoma of the small intestine. These abnormalities may ormay not reflect the effect of irradiation. In contrast, the singlesurvivor in the antibiotics only group had focal typhlitis andperforation associated with peritonitis. Ionizing radiation inducesdisruption of the mucosal integrity which is often complicated byulceration (26, 36). Focal ulcerations are common; these vary fromsimple loss of epithelial layer with acute inflammation of the laminapropria to ulcers that may penetrate to varying depths of the intestinalwall, even to the serosa. A perforated appendix and associatedperitonitis is a frequent clinical consequence of exposure to ionizingradiation in patients (26). It was concluded that the cecal perforationwas a result of radiation injury, and the mouse survived until the endof the study due to antibiotic administration, which prevented fatalperitonitis.

The ideal radioprotective agent would both be protective andcost-effective. Black edible mushrooms, in their native form, provide anatural radioprotector that is readily available. The equal survival ofmice protected with either black mushrooms or white mushroomssupplemented with melanin establishes the causality between the presenceof melanin in black mushrooms and their radioprotective properties.Interestingly, approximately the same percentage of mice survived inexperiments with mushrooms when no antibiotics were given as in theexperiment with the synthetic pheomelanin where the supplementation withantibiotics was required for the protection. This effect is most likelydue to the combination of melanin and soluble antioxidants which arepresent in mushrooms (FIG. 2). Given that a significant proportion ofblack mushroom- or white mushroom-supplemented with melanin-fed micebecame long term survivors it must follow that the presence of melaninin the GI tract provided local protection that allowed these mice torecover. Protection of GI mucosa would prevent death by a GI syndromeand sepsis. Hence, local GI protection appears to translate intosystemic protection and this observation establishes a new concept inthe approach to protecting against radiation sickness. Black ediblemushrooms could be prepared as a suspension in a palatable liquid anddistributed as food supplement to affected populations. Thisradioprotection may also benefit cancer patients undergoing radiationtreatment, as radiation-induced injury to the GI tract is common inpatients undergoing external radiation beam therapy (EBRT).

Materials and Methods

Melanin sources and physico-chemical analyses. Commercial syntheticeumelanin made from tyrosine was obtained from Sigma-Aldrich. Themicrobial eumelanin from C. neoformans strain 24067 in form of “ghosts”(hollow melanin spheres from which all cellular contents has beenremoved via multi-step purification procedure) was purified aspreviously described8. Synthetic pheomelanin using 5-S-cysteinyldopa wasproduced by incubating 0.5 mmol 5-S-cysteinyldopa with 0.025 mmolL-DOPA, added as a catalyst, in 0.05 M sodium phosphate buffer, pH 6.8and with mushroom tyrosinase (Sigma) in the amount of 8300 units (773 μLof 2 mg/mL solution) with constant agitation overnight at 37° C. Afterthe overnight incubation, the oxidation reaction was halted by theaddition of 250 μL 6 M HCl to lower the pH to approximately 3.0. Thisacidified mixture was kept at 2° C. for 1 hour. The precipitate wascollected by centrifugation, washed three times with 15 mL 1% aceticacid, washed twice with 15 mL acetone, once more with 15 mL 1% aceticacid, and re-suspended in de-ionized water. The resulting pheomelaninwas then lyophilized and suspended in PBS at a concentration of 12.5mg/mL to create the stock suspension.

Dried Auricularia auricula-judae (black mushrooms) and Boletus edulis(white mushrooms) were purchased from Trader Joe's (Monrovia, Calif.).Melanin from black mushrooms was purified as described previously (19).Elemental analysis of melanin was carried out by QTI (Whitehouse, N.J.).EPR of dried mushrooms and oxidative HPLC of melanin using permanganateoxidation were performed as in (18). The antioxidant capacity ofmethanol extracts from black and white mushrooms in DPPH assay wasmeasured as in (23).

Evaluation of potential toxicity of melanins. All animal studies werecarried out in accordance with the guidelines of the Albert EinsteinCollege of Medicine Animal Care and Use Committee. Six-eight weeks oldCD-1 female mice (Charles River Breeding Laboratories, Portage, Mich.)were used in all experiments. Mice were divided into groups of five andfed 15 mg/kg body weight either synthetic or microbial eumelanins, or100 mg/kg synthetic pheomelanin or PBS via gavage needle. Mice wereevaluated daily for body weight and their physical condition. Two miceper group were humanely sacrificed at 24 hr post-feeding with melanin,and the remaining mice were sacrificed at day 14. The stomach and smalland large intestines were fixed in 10% neutral buffered formalin androutinely processed for paraffin embedding. Samples were sectioned to 5μm and stained with hematoxylin and eosin (H&E) for histologicalevaluation.

Imaging. The in vivo imaging was performed with IVIS Spectrum ImagingSystem (Caliper Life Sciences, Hopton, Mass.) in epifluorescence modeequipped with 675/30 nm and 840/20 nm filters for excitation andemission, respectively. Mice were fed with non-fluorescent chow for 5days and then fasted overnight before the imaging experiment to excludethe interference from the remnant autofluorescence of the chow. Theywere given 1 g/kg body weight white mushrooms suspension in water viagavage needle and imaged in supine position under Isoflurane anesthesiaat 15, 30 and 60 min post-feeding.

In vivo radioprotection with various melanins. Microbial and syntheticeumelanins. CD-1 mice (13 mice per group) were fed either syntheticeumelanin or microbial eumelanin or PBS via gavage needle at a dose of15 mg/kg body weight. One hour post-eumelanin feeding the mice weresubjected to whole body irradiation in a 137-Cs irradiator with a totalbody dose of 9 Gy delivered at 2.5 Gy/min. At 4 and 24 hr, 2 mice pergroup were humanely sacrificed and their stomachs, small intestine andcolon were removed and processed as previously described. The remaininganimals were monitored until death with daily measurements of bodyweight. Moribund animals were humanely euthanized.

Synthetic pheomelanin. CD-1 mice were divided into 5 groups of fifteenmice. The groups were treated with 0, 25, 50, 75, 100 mg/kg melaninsuspension in PBS via gavage. One hr post feeding the mice weresubjected to whole body irradiation in a 137-Cs irradiator with a totalbody dose of 9 Gy delivered at 2.5 Gy/min and their body weight andsurvival were monitored for 40 days. The rate of weight change wasquantified by using a linear regression analysis (Prism, GraphPad, SanDiego, Calif.). In a follow-up study mice were divided into threegroups. Group 1 was treated by oral gavage with 100 mg/kg melaninsuspension in PBS and group 2 and 3 were treated by oral gavage withonly PBS followed by irradiation as above of all three groups. Startingat 2 days after irradiation groups 1 and 2 were dosed subcutaneouslywith penicillin (10,000 units/mL) and streptomycin (10 mg/mL) (Sigma,St. Louis, Mo.) at 120 μL twice a day for 5 days. At the completion ofthe study on day 40, all surviving mice were sacrificed and theirstomachs, small intestine, large intestine, liver, sternum and femurwere removed and processed as previously described for histologicalevaluation.

Black mushrooms. Since dried black mushroom contain 10% melanin, blackmushrooms were administered as suspension in sterile PBS via gavage as 1g/kg body weight dose to match the melanin concentrations in thedescribed above experiments with pure synthetic melanins. CD-1 mice weredivided into groups of 5-6 and fed 1 g/kg body weight black mushroomsuspension in PBS, or PBS alone, or 1 g/kg white mushroom suspension, or1 g/kg white mushroom suspension supplemented with 100 mg/kg syntheticmelanin via gavage needle. One hour after mushroom administration micewere irradiated with 9 Gy dose of Cs-137 radiation at a dose rate of 2.5Gy/min. Mice were evaluated daily for body weight and their physicalcondition for 45 days. The experiment was performed twice. At theconclusion of the experiment the surviving mice were humanelysacrificed, their blood chemistry was analyzed for white blood cells andplatelet count, gross pathology was performed and the stomach, small andlarge intestines, spleen and bone marrow were subjected to histologicalevaluation. Survival of mice was analyzed using log-rank test, the WBCand platelet counts—by one tail Student's test. The differences inresults were considered statistically significant when P was <0.05.

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1. A method for alleviating and/or preventing one or more side effectsassociated with exposure to radiation in a subject exposed to radiationor at risk for exposure to radiation comprising oral administration tothe subject of an amount of an edible source of melanin effective toalleviate a side effect associated with radiation.
 2. A method forincreasing the survival rate of a plurality of subjects exposed to anamount of radiation likely to kill the plurality of subjects, comprisingoral administration to each of the plurality of subjects of an amount ofan edible source of melanin effective to increase the survival rate ofthe plurality of subjects exposed to the amount of radiation likely tokill the plurality of subjects.
 3. The method of claim 1, whereinmelanin is administered to the subject, or to the subjects, in an amountequivalent to at least 8 mg of purified melanin per kg of body weight ofthe subject.
 4. The method of claim 1, wherein the melanin is isolatedor derived from a melanin-containing biological source where melaninconstitutes at least 10% of the dry weight of the biological source, ormelanin is provided by administering a melanin-containing biologicalsource that comprises at least 10% melanin per dry weight of thebiological source.
 5. The method of claim 4, wherein the biologicalsource is a melanin-containing plant, cell, fungus or microorganism. 6.The method of claim 4, wherein the biological source is grown in thepresence of a melanin precursor.
 7. The method of claim 6, wherein themelanin precursor is one or more of L-dopa (3,4-dihydroxyphenylalanin),D-dopa, catechol, 5-hydroxyindole, tyramine, dopamine, tyrosine,cysteine, m-aminophenol, o-aminophenol, p-aminophenol, 4-aminocatechol,2-hydroxyl-1,4-naphthaquinone, 4-metholcatechol, 3,4-dihydroxynaphhalene, gallic acid, resorcinol, 2-chloroaniline, p-chloroanisole,2-amino-p-cresol, 4,5-dihydroxynaphthalene, 1,8-dihydroxynaphthalene,2,7-disulfonic acid, o-cresol, m-cresol, and p-cresol.
 8. The method ofclaim 5, wherein the fungus is a melanin-containing mushroom.
 9. Themethod of claim 8, wherein the fungus is Auricularia auricular-judae.10. The method of claim 3, wherein melanin is administered in an ediblesubstance of at least 80 mg of edible substance per kg of body weight ofthe subject.
 11. The method of claim 10, wherein melanin is administeredas at least 80 mg of dry mushrooms per kg of body weight of the subject.12. The method of claim 1, wherein an internal organ of the subject isprotected from radiation.
 13. The method of claim 12, wherein the organthat is protected is one or more organ selected from the groupconsisting of bone marrow, liver, spleen, kidneys, lungs, andgastrointestinal tract.
 14. The method of claim 1, wherein the sideeffect associated with radiation is one or more of acute-nature nausea,vomiting, abdominal pain, diarrhea, dizziness, headache, fever,cutaneous radiation syndrome, low blood cell count, infection due to lowwhite blood cells, bleeding due to low platelets, anemia due to low redblood cells, or death. 15-19. (canceled)
 20. The method of claim 1,wherein the source of the radiation is radiotherapy used for treatmentof disease, radiation from a medical imaging device, radiation used forradiation surgery, a nuclear weapon, a nuclear reactor, high-altituderadiation.
 21. The method of claim 20, wherein the source of radiationis from a nuclear power plant.
 22. The method of claim 1, wherein theedible source of melanin comprises a drinkable suspension of melaninpackaged for oral administration to a subject for alleviating and/orpreventing one or more side effects associated with exposure of thesubject to radiation, wherein the drinkable suspension comprises atleast 500 mg melanin in a volume of at least 10 mL.
 23. The method ofclaim 1, further comprising administering one or more antibiotics to thesubject or to the subjects.
 24. An edible source of melanin packaged fororal administration to a subject for alleviating and/or preventing oneor more side effects associated with exposure of the subject toradiation, wherein the edible source provides melanin in an amountequivalent to at least 8 mg of purified melanin per kg of body weight ofthe subject or a drinkable suspension of melanin packaged for oraladministration to a subject for alleviating and/or preventing one ormore side effects associated with exposure of the subject to radiation,wherein the drinkable suspension comprises at least 500 mg melanin in avolume of at least 10 mL.
 25. (canceled)
 26. The drinkable suspension ofclaim 2, wherein the drinkable suspension comprises at least 500 mgmelanin in a volume of at least 100 mL.